Polymer-bound camptothecin derivatives

ABSTRACT

A polymeric conjugate consists essentially of: (i) from 60 to 99 mol % of N-(2-hydroxypropyl) methacryloylamide units represented by formula 1: ##STR1## (ii) from 1 to 40 mol % of 20-0-(N-methacryloylglycyl-aminoacyl) camptothecin units represented by formula 2 ##STR2## wherein  A! is a spacer group having respective terminal amino and carbonyl groups which are separated by at least three atoms and O-CPT represents a residue of a camptothecin, the C-20 hydroxy group of the camptothecin being linked to the terminal carbonyl group of the spacer group  A!; and (iii) from 0 to 10 mol % of N-methacryloylglycine or N-(2-hydroxy-propyl)methacryloylglycinamide units represented by formula 3: ##STR3## wherein Z represents hydroxy or a residue of formula --NH--CH 2  --CH(OH)--CH 3 .

This application is a 371 of PCT/EP94/03154 filed Sep. 21, 1994.

The present invention refers to water soluble polymer-bound camptothecinand polymer-bound camptothecin derivatives endowed with antitumouractivity, to a process for their preparation and to pharmaceuticalcompositions containing them.

Camptothecin is an alkaloid isolated from the leaves and bark ofCamptotheca acuminata; other analogs of camptothecin are also known andwere prepared by semisynthesis from camptothecin or by total synthesis:see J.Amer.Chem.Soc. 94(10), 3631 (1972); J.Chem.Soc.D. (7), 404 (1970);U.S. Pat. No. 4,981,969 (Jan. 1, 1991); U.S. Pat. No. 5,049,668 (Sep.17,1991).

Camptotnecin has a pentacyclic structure consisting of a fused ringsystem forming a quinoline ring (rings A and B), a pyrrolidine ring(ring C), a pyridone ring (ring D) and an α-hydroxy-δ-lactone moiety(ring E). Camptothecin and several of its A ring-substituted derivativesexhibit antitumour activity against a variety of solid tumour lines,including cell lines resistant to clinically available chemotherapeuticagents see: J.Clin.Pharmacol. 30, 770 (1990); CancerChemother.Pharmacol. 28, 192 (1991)!.

Camptothecin, as well as most of its derivatives, is practicallyinsoluble in vehicles suitable for parenteral administration due to weakbasicity of the quinone nitrogen atom. In order to solubilizecamptothecins, several water soluble prodrugs have been proposed such as20-O-phosphate or 20-O-acylamino derivatives which can be protonated bymineral acids, thus allowing solubility: see U.S. Pat. No. 4,943,579(Jul. 24, 1990). Toxic side effects, including haematological andgastrointestinal ones, are associated with the administration of thesedrugs. Numerous attempts have been made to improve therapeutic index ofcamptothecin by modifying its structure.

The present invention provides polymeric conjugates of camptothecinswhich are water soluble and possess antitumor activity in vivo anddecreased toxicity. More particularly, the invention provides apolymeric conjugate which is denoted herein as A and which consistsessentially of:

(i) from 60 to 99 mol % of N-(2-hydroxypropyl) methacryloylamide unitsrepresented by formula 1: ##STR4## (ii) from 1 to 40 mol % of20-O-(N-methacryloylglyl aminoacyl)camptothecin units represented byformula 2 ##STR5## wherein A! is a spacer group having respectiveterminal amino and carbonyl groups which are separated by at least threeatoms and O-CPT represents a residue of a camptothecin, the C-20 groupof the camptothecin being linked to the terminal carbonyl group of thespacer group A!; and (iii) from 0 to 10 mol % of N-methacryloylglycineor N-(2-hydroxy-propyl) methacryloylglycinamide units represented byformula 3: ##STR6## wherein Z represents hydroxy or a residue of formula--NH--CH₂ --CH(OH)--CH₃.

The invention also provides a process for preparing a polymericconjugate as defined above, which process comprises reacting a20-O-acylamino-camptothecin derivative of formula 7:

    H A!--O--CPT                                               7

wherein A! and O-CPT are as defined above, with a polymer consistingessentially of:

(i) from 60 to 99 mol % of N-(2-hydroxypropyl)methacryloyl-amide unitsrepresented by formula 1: ##STR7## and (iv) from 40 to 1 mol % ofN-methyacryloylglycine units represented by formula 4: ##STR8## whereinR₂ is (a) the residue of an active ester or (b) hydroxy; and optionallydisplacing the remaining active ester groups with 1-amino-2-propanol.

The polymeric conjugate A contains the N-(2-hydroxypropyl)methacryloylamide units represented by the formula 1 in a proportion of60 mol % or more, for example at least 80 mol % or at least 85 mol %.These units may be present in an amount from 91 to 98 mol %. Theconjugate may also contain from 1 to 40 mol % of the 20-O-(Nmethacryloylglycyl-aminoacyl)camptothecin units represented by theformula 2, for example from 1 to 20 mol % of such units. The conjugatemay contain from 1 to 8 mol %, for example from 2 to 6 mol %, of theseunits.

The spacer group 8A! may be from three to twelve, for example from sixto nine, atoms long. Typically, the group is susceptible tointracellular hydrolysis. Preferably it is resistant to extracelluarhydrolysis. The spacer group may be a peptide spacer, for example from 1to 4 or 2 to 4 amino acid residues long. The spacer may thus be adipeptide, peptide or tetrapeptide.

Preferably the spacer group A! is selected from Ala-Gly, Phe-Gly,Phe-Phe, Leu-Gly, Val-Ala, Phe-Ala, Leu-Phe, Leu-Ala, Phe-Leu-Gly,Phe-Phe-Leu, Leu-Leu-Gly, Phe-Tyr-Ala, Phe-Gly-Phe, Phe-Phe-Gly andPhe-Leu-Gly-Phe. Alternatively the spacer group A! is a group offormula--HN-Y-CO- in which Y is C₃ -C₆ linear or branched alkyl such as--(CH₂)_(n) -- wherein n is 3, 4, 5 or 6.

Alternatively, the spacer A! is a group of formula Ala-Gly-NH-Y-CO-,Phe-Gly-NH-Y-CO-, Phe-Phe-NH-Y-CO-, Leu-Gly-NH-Y-CO-, Val-Ala-NH-Y-CO-,Phe-Ala-NH-Y-CO-, Leu-Phe-NH-Y-CO-, Leu-Ala-NH-Y-CO-,Phe-Leu-Gly-NH-Y-Co-, Phe-Phe-Leu-NH-Y-CO-, Leu-Leu-Gly-NH-Y-CO-,Phe-Tyr-Ala-NH-Y-CO, Phe-Gly-Phe-NH-Y-CO-, Phe-Phe-Gly-NH-Y-CO- orPhe-Leu-Gly-Phe-NH-Y-CO- wherein Y has the same meaning as above.

A residue of a camptothecin is denoted by O-CPT. The camptothecin may becamptothecin itself or an analogue is such as an A-ring analogue. Suchan A-ring analogue is thus camptothecin substituted on the A-ring. Thecamptothecin may be in the natural S form or in the R form or in amixture of R and S forms (racemic mixture). Suitable camptothecinresidues O-CPT are denoted by the formula 5: ##STR9## wherein R₁represents, for example, hydrogen, hydroxy, nitro or amino or amethylenedioxy group bonded to two adjacent carbon atoms on the A-ring.Preferably R₁ represents hydrogen, 9-, 10-, 11- or 12-hydroxy, 9- or10-nitro, 9- or 10-amino or 10,11-methylenedioxy. More preferred arecamptothecin residues of formula 6: ##STR10## wherein R₁ is as definedabove.

Preferably, the units of formula 2 are present in an amount of from 1 to10 mol % such as 0.5 to 5 mol %. Also preferably, the camptothecincontent is from 1 to 10 % (w/w), more preferably from 4 to 8 % (w/w),based on the polymeric conjugate.

The invention also provides a 20-O-acylamino-camptothecin derivative offormula 7 as defined above. The present invention further provides aprocess for preparing a 20-O-acylamino-camptothecin derivative offormula 7, which process comprises condensing a camptothecin with aN-protected aminoacyl derivative of formula 8:

    R.sub.3 -- A!--P                                           8

wherein A! is as defined above and R₃ represents an amino-protectinggroup, such as t-boc, CBZ, FMOC, triphenylsilyl, diphenylmethylene ortriphenylmethyl, and P is a residue of an activated ester, such asp-nitrophenoxy, pentafluorophenoxy or N-hydroxysuccinimido, in thepresence of an activating agent such as 4-dimethylaminopyridine, to givea N-protected-20-O(acylamino) compound represented by formula 9:

    R.sub.3 -- A!-- O--CPT!

wherein R₃, A! and O-CPT! are as defined above; and removing theN-protecting group from the resulting compound.

Preparation of compounds of formula 8 follows standard syntheticprocedures that are known from the literature. SuitableN-protected-aminoacyl derivatives of formula 8 includeN-trityl-L-phenylalanyl-L-leucyl-glycyl p-nitrophenyl ester (8a) orN-trityl-glycyl-L-leucyl-glycyl p-nitrophenylester (8b), or6-N-trityl-hexa-noyl p-nitrophenyl ester (8c). Some derivatives offormula 8 and their preparation are described also in our copendingInternational Patent Application No. PCT/EP94/01100.

Thus, for example, a camptothecin may be allowed to react with a molarexcess, for example up to a five-fold molar excess or more, especially 2mol. equivalent, of a N-protected-aminoacyl derivative of formula 5 inanhydrous solvent such as anhydrous dimethylformamide ordimethylsulfoxide in the presence of activating agent such as4-dimethylaminopyridine. In this manner, the protected amino acid isintroduced at position C-20 on the camptothecin molecule. The reactioncan typically be effected for from 8 to 24 hours. The reaction istypically carried out at a temperature from 15° to 40° C. It should benoted that, following such reaction conditions, 9-aminocamptothecinregiospecifically reacts at the C-20-hydroxy position due the weakbasicity of the 9-amino group.

The amino-protecting group R₃ is removed by an appropriate deprotectingagent to give the 20-O-acylamino-camptothecin derivative of formula 7.Deprotection may therefore be achieved by mild acid treatment, such astreatment with acetic acid, or by reduction. Hydrogenolysis maytherefore be employed.

The condensation of the 20-O-acylamino-camptothecin derivative offormula 7 with the polymer consisting essentially of from 60 to 99 mol %of the N-(2-hydroxypropyl)-methacryloylamide units of formula 1 and from40 to 1 mol % of N-methacryloylamide units of formula 4, and theoptional subsequent displacement of the remaining active ester groups,are carried out in conditions capable of preserving the nature oflinkage between camptothecins and spacers A! as well as that of theconjugate.

Polymers consisting essentially of from 60 to 99 mol % of theN-(2-hydroxypropyl)-methacryloylamide units of formula 1 and from 40 to1 mol % of N-methacryloylglycine units of formula 4, are prepared bycopolymerization of N-(2- hydroxypropyl)methacrylamide withN-methacryloyl-glycine or N-methacryloylglycine active-esterderivatives, as described in Makromol.Chem. 178, 2159 (1977). R₂ mayrepresent a phenyloxy group which is substituted on the phenyl ring byone or more electron-withdrawing groups. Examples of suitableelectron-withdrawing groups include nitro (--NO₂) and halogen. R₂ ispreferably the leaving group: ##STR11## wherein L is an electronwithdrawing group, for example --NO₂ or a halogen such as fluorine orchlorine, and m is an integer of 1 to 5, typically 1 to 3, preferably 1or 2. Preferably R₂ is a p-nitrophenoxy group or a 2,4-dichlorophenoxygroup.

Preferably, the reaction between a polymer in which R₂ represents (a)the residue of active ester and a compound of formula 7 to prepare thepolymer conjugate of the invention is carried out in an anhydrous polarorganic solvent such as dimethyl-formamide or dimethylsulfoxide. Thereaction can typically be effected for from 8 to 24 hours. The reactionis typically carried out at temperature from 15° to 30° C., preferablyat room temperature for 15 hours; then the aminolysis of the remainingactive ester can be performed in the presence of 1-amino-2-propanol atroom temperature, for from 0.5 to 1 hour. The conjugate suitably isprecipitated with acetone, dissolved in ethanol and reprecipitated withacetone.

In another method, in order to prepare a polymer conjugate of theinvention, the reaction between a polymer in which R₂ represents hydroxygroup (b) and a compound of formula 7 is carried out in an anhydrouspolar solvent such as dimethylformamide or dimethylsulfoxide in thepresence of a condensing agent such as2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline. The reaction cantypically be effected for from 8 to 24 hours. The reaction is typicallycarried out at a temperature from 15° to 30° C., preferably at roomtemperature for 15 hours; then the conjugate can be precipitated withacetone, dissolved in ethanol and reprecipitated with acetone.

For example, the polymer in which R₂ represents (a) the residue of anactive ester, provided at a concentration of 15% (weight/volume) inanhydrous dimethylsulfoxide, is treated with a 20-O-aminoacylcamptothecin derivative of formula 7, 3% (w/v), at room temperature for15 hours. Then 1-amino-2-propanol, 0.1% (w/v) is added and the reactionmixture is kept at room temperature for 1 hour. The conjugate can beprecipitated with acetone, then dissolved with absolute ethanol at aconcentration of 10% (w/v) and precipitated again with acetone to giveneutral camptothecin conjugate according to the invention.

In another example, the polymer in which R₂ represents (b) hydroxy,provided at a concentration of 15% (weight/volume) in anhydrousdimethylsulfoxide, is treated with a 20-O-aminoacyl camptothecinderivative of formula 7, 3% (w/v), in the presence of2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 1.3% (w/v), at roomtemperature for 15 hours. Acetone then is added to cause precipitation,the precipitate is dissolved with absolute ethanol at a concentration of10% (w/v) and precipitation is again caused by acetone addition to givea polymeric conjugate according to the invention.

The content of active drug, such as camptothecin or its A-ringanalogues, in polymeric conjugates of the invention is determined byHPLC or absorbance spectroscopy analysis.

Polymer conjugates of camptothecin and its A-ring analogues described inthe present invention are endowed with improved water solubility anddecreased toxicity. The conjugates exhibit good water solubility,biocompatibility and release camptothecins in the plasma or afterinternalization into cells by enzymatic cleavage.

BIOLOGICAL ACTIVITY

Copolymer of N-(2-hydroxypropyl)methacrylamide, 20-O-N-methacryloylglycyl -L-phenylalanyl-L-leucylglycyl! camptothecin andN-(2-hydroxypropyl) methacryloylglycinamide (A2) was tested in nude micetransplanted with HT29/Colon Carcinoma (Table 1) and in mice bearingearly and advanced M5076 murine reticulosarcoma (Table 2 and 3) incomparison with free camptothecin (CPT).

When compared with camptothecin, a striking higher activity in allexperiments was observed for the polymer bound camptothecin derivativeA2. It is noteworthy that cured mice were found in the experiment onHT29/Colon Carcinoma.

The antitumor activity was tested with the same treatment schedule forA2 and CPT.

It should be noted that polymer-bound camptothecin A2 and was foundhighly water soluble and was dissolved in saline and was administeredi.v.; whereas CPT was dissolved in a mixture of water and Tween 80.

Drug preparation and administration

Compound A2 was dissolved in distilled water immediately prior to useand the concentration was checked spectrophoto-metrically at 370 nm El %57.18). Camptothecin (CPT) was dissolved in sterile water with 10%tween.

Treatment was administered i.v. in a volume of 10 ml/kg of body weightand control mice were treated with sterile water.

Tumors

HT29 Colon Carcinoma was transplanted s.c. in athymic Swiss/nu/ miceusing 15-20 mg of tumor brei.

M5076 muine reticulosarcoma was maintained by serial i.m. passages andtransplanted s.c. (5×10⁵ cells/mouse) in compatible C57Bl/6 mice.

All animals were from Charles River Calco, Como, Italy.

Ten mice/group for conventional and eight for athymic mice were used.

The conventional mice weighed 20 to 22 g and were kept under standardlaboratory conditions.

The mouse colony was routinely tested for the absence of antibodies to apanel of pathogens including Mouse Hepatitis, Sendai Virus andMycoplasma Pulmonis.

Evaluation of antitumor activity and toxicity

Tumor growth was assesed by caliper measurement and the tumor weightestimated according to Geran et al. (see: Cancer Chem.Rep., Part 3, 3(2) ed. 3, pp 1-103, 1972). The antitumor activity is expressed aspercentage of inhibition of tumor growth (%T/I) using the followingformula: ##EQU1## The median increase in survival time (T/C%) wascalculated using the following formula: ##EQU2##

Cured mice are mice without tumor at the end of the experiment.

Toxicity was evaluated on the basis of the body weight reduction andgross autopsy findings, mainly in terms of reduction of spleen and liversize.

                  TABLE 1                                                         ______________________________________                                        Antitumor Activity of Compound A2 in comparison                               with Camptothecin (CPT) against HT29/Colon Carcinoma.                                 dose.sup.(1)                                                                           treatment                                                                              % tumor       cured.sup.(3)                         Compound                                                                              mg/kg    schedule inhib.  TOX.sup.(2)                                                                         mice                                  ______________________________________                                        A2      7.5      iv q4dx6 96      0/18  3/18                                  CPT     7.5      iv q4dx6 83      0/10  0/10                                  ______________________________________                                         .sup.(1) expressed as CPT equivalent.                                         .sup.(2) number of toxic deaths/total number of mice                          .sup.(3) tumor free mice 90 days after tumor implant                     

                  TABLE 2                                                         ______________________________________                                        Antitumor Activity of Compound A2 in comparison                               with Camptothecin (CPT) against early M5076 murine                            reticulosarcoma.                                                                       dose.sup.(1)                                                                          treatment % tumor.sup.(3)                                                                      T/C                                         Compound mg/kg   schedule  inhib. %     TOX.sup.(2)                           ______________________________________                                        A2       7.5     iv 1,6,9  100    171   0/10                                  CPT      7.5     ip 1,6,9  100    165   0/10                                  ______________________________________                                         .sup.(1) expressed as CPT equivalent.                                         .sup.(2) number of toxic deaths/total number of mice                          .sup.(3) % tumor inhibition was estimated one week after the last             treatment.                                                               

                  TABLE 3                                                         ______________________________________                                        Antitumor Activity of Compound A2 in comparison                               with Camptothecin (CPT) against advanced M5076 murine                         reticulosarcoma. Treatment schedule iv on day 16,20,24                        28,31,35.                                                                                Dose.sup.(1)                                                                          % tumor.sup.(3)                                                                           T/C                                            Compound   mg/kg   inhibition  %    TOX.sup.(2)                               ______________________________________                                        A2         10.0    80          174  0/10                                                 15.0    95          183  0/10                                      CPT        7.5     72          173  0/10                                      ______________________________________                                         .sup.(1) expressed as CPT equivalent.                                         .sup.(2) number of toxic deaths/total number of mice                          .sup.(3) % tumor inhibition was estimated at day 34.                     

Toxicity

Toxicity of copolymer of N-(2-hydroxypropyl)methacryl-amide, 20-O-N-methacryloylglycyl-L-phenylalanyl-L-leucylglycyl ! camptothecin andN-(2-hydroxypropyl) methacryloylglycinamide (A2) was evaluated inhealthy C57Bl/F mice, treatment i.v. in comparison with camptothecin(CPT).

    ______________________________________                                        The LD.sub.10.sup.(1) and LD.sub.50.sup.(2) values in C57B1/F mice are as     follows:                                                                                       LD.sub.10                                                                             LD.sub.50                                            Compound         mg/kg   mg/kg                                                ______________________________________                                        A2               129     151                                                  CPT              16.9    43.4                                                 ______________________________________                                         .sup.(1) LD.sub.10 : dose inducing 10% of death mice.                         .sup.(2) LD.sub.50 : dose inducing 50% of death mice.                    

The low toxicity of polymer-bound camptothecin derivative A2

allows to administer higher doses of product and to reach equivalent orbetter results than that with camptothecin.

The polymer-bound camptothecins have anti-tumor activity. They inhibittopoisomerase I. They are useful in the treatment of leukaemia and colonand rectal tumors in particular.

A human or animal can therefore be treated by a method comprisingadministering thereto a therapeutically effective amount of a polymericconjugate of the invention. The condition of the human or animal patientcan thus be improved.

The dosage range adopted will depend on the route of administration andon the age, weight and condition of the patient being treated. Thepolymeric conjugates are typically administered by the parenteral route,for example intramuscularly, intravenously or by bolus infusion. Asuitable dose range is from 1 to 1000 mg of camptothecin equivalent perm² body surface area, for instance from 10 to 50 mg/m².

The polymeric conjugates may be formulated into a pharmaceuticalcomposition together with a pharmaceutically acceptable carrier ordiluent. Typically the polymeric conjugates are formulated forparenteral administration, for example by dissolution in water forinjection or physiological saline.

The following Examples illustrate the invention without limiting it.Throughout the specification, amino acid residues are shown in thethree-letter code according to Eur.J.Biochem. 138, 9-37, 1984.

Stability of polymeric conjugates in murine or human plasma was assessedin the following manner: to 1 ml of murine or human plasmas, variousconcentrations of a polymeric conjugate of the invention A were addedand at appropriate time (24, 48, 72, 96 hours) 100 μl samples werecollected and stored at -70° C. until further processing.

Each sample was extracted by adding 100 μl of 0.25M phosphoric acid, 500μl CH₃ CN and 700 μl ethyl acetate and vigorously shaking for 20 minutesat 4° C. After that time, the sample was spun at 15000×g for 10 minutesand the supernatant was separated. To the residue was added 300 μl ofCH₃ CN and 500 μl was spun at 15000×g for 10 minutes. The supernatantwas separated. The organic phases were pooled and evaporated using ahigh vacuum centrifuge. The sample was recovered by adding 500 μl ofMeOH/water pH2 (70/30 by volume) and injected into HPLC apparatus fordetermining the total drug percentage content.

    ______________________________________                                        HPLC system                                                                   ______________________________________                                        Column     μBondapak C10 (Waters) 3.9 × 300 mm                       Flow rate  1.5 ml/min                                                         Detector   Spectrophotometer Fluorescence 650-40                                         (Perkin Elmer); emission 434 nm (slit 5);                                     excitation 370 nm (slit 5)                                         Injection  10 μl                                                           Mobile Phase                                                                             51.5% MeOH, 47.5% water, 1% phosphoric acid                        ______________________________________                                    

Example 1

6-N-tritvl-hexanoyl p-nitrophenyl ester (8c)

    (C.sub.6 H.sub.5).sub.3 C--NH(CH.sub.2).sub.5 CO--OC.sub.6 H.sub.4 pNO.sub.2                                                 8c

6-Aminocaproic acid (6.5 g, 50 mmol) suspended in a mixture of drychloroform (75 ml) and dry acetonitrile (15 ml) was added withtrimethylsilyl chloride (6.3 ml, 50 mmol) and kept under reflux for twohours under vigorous stirring. After that, the mixture was cooled andadded in sequence were triethylamine (13.7 ml, 100 mmol) and tritylchloride (14 g, 50 mmol) dissolved in dry chloroform (100 ml). Themixture was let to stand overnight at room temperature, then methanol(10 ml) was added and the reaction mixture was concentrated underreduced pressure. The residue was picked up with cold aqueous 5% citricacid (200 ml) and extracted with ethyl acetate (200 ml). The organicphase was separated and extracted with cold aqueous 1N sodium hydroxide(200ml). The aqueous phase was separated, cooled with ice, brought to pH5 with acetic acid and extracted with ethyl acetate (2×100 ml). Theorganic solvent was removed under reduced pressure to afford, aftercrystallization from ethyl acetate, 6-N-trityl-hexanoic acid (16 g).This material was dissolved in anhydrous tetrahydrofurane (150 ml) andtreated with p-nitrophenol (5.6 g, 400 mmol) anddicyclohexylcarbodiimide (8.4 g, 40 mmol) at 0° C. for one hour understirring, then overnight at 8° C. After that, the reaction mixture wasfiltered, cooled at 0° C. for two hours and filtered again. Finally, theorganic solvent was removed under reduced pressure and the residue wascrystallized with ethyl ether to afford the title compound 8c (16.4 g).TLC on Kieselgel plates F₂₄₅, (Merck), eluting system ethylether/n-hexane (1:1 by volume) R_(f) =0.6; FD-MS: m/z M+H!⁺ 495 H¹ NMR(90 MHz, CDCl₃) δ: 1.1-1.9 (m, 6H); 2-2.5 (m, 4H); 5.7-5.9 (m, 2H, NH,--COOH); 7.2-7.7 (m, 15 H).

Example 2

20-O-(6-aminohexanoyl)camptothecin (7a) ##STR12##

Camptothecin (6a, R₁ =H, 0.7 g, 2 mmol) was dissolved in drydimethylsulfoxide (100 ml) and added with 6-N-trityl-hexanoylp-nitrophenyl ester (8c, 2 g, 4 mmol), prepared as described in Example1, and 4-dimethylaminopyridine (0.24 g, 2 mmol). The reaction mixturewas kept at room temperature for 48 hours, then diluted with chloroform(400 ml) and washed with water (3×100 ml). The organic phase wasseparated, dried over anhydrous sodium sulphate and concentrated tosmall volume under reduced pressure. The crude material was flashchromatographed on silicic acid column eluting with chloroform to afford20-O-(6-N-trityl-hexanoyl)camptothecin (0.6 g). TLC on Kieselgel platesF₂₄₅ (Merck), eluting system chloroform/methanol (95:5 by volume) R_(f)=0.4. The N-protected derivative was treated with 95% trifluoroaceticacid (10 ml) at room temperature for 50 minutes. After removal of theacid under reduced pressure, the title compound 7c was collected withethyl ether. Yield 0.4 g. TLC on Kieselgel plates F₂₄₅ (Merck), elutingsystem methylene chloride/methanol/acetic acid/water (80:20:7:3) R_(f)=0.6. FD-MS: m/z M+H!⁺ 462

Example 3

20-O-(L-Phenylalanyl-L-Leucyl-Glycl)camptothecin (7b) ##STR13##

N-trityl-L-phenylalanyl-L-leucylglycine p-nitrophenyl ester (8a, 1.4 g,2 mmol) prepared as previously described in UK Application No.9309663.4, camptothecin (6a, R₁ =H, 0.35 g, 1 mmol) and4-dimethylaminopyridine (0.12 g, 1 mmol) were dissolved with drydimethylsulfoxide (50 ml) and stirred at room temperature for 20 hours.After that, the reaction mixture was diluted with chloroform (400 ml)and washed with water (3×100 ml). The organic phase was separated, driedover anhydrous sodium sulphate and concentrated to small volume underreduced pressure. The crude material was flash chromatographed onsilicic acid column eluting with a mixture of chloroform/methanol(99.5/0.5 by volume). The fractions containing the N-protectedderivative of the title compound were pooled, concentrated to dryness,dissolved with aqueous 75% acetic acid (30 ml) and kept at roomtemperature for one hour. The reaction mixture was treated wtih solidsodium hydrogen carbonate to pH 7 and extracted with chloroform (400ml). After removal of the organic solvent, the title compound 7b wascrystallized from ethyl ether. Yield 0.16 g. TCL on Kieselgel platesF₂₄₅ (Merck), eluting system chloroform/methanol (9:1 by volume) R_(f)0.35

FD-MS:m/z M+H!⁺ 667

¹ H-NMR (400 MHz,CDCl₃) δ:

0.81 (d, J=6.5Hz, 3H, δ-Leu); 0.82 (d, J=6.6 Hz, 3H, δ'-Leu);

0.98 (t, J=7.6 Hz, 3H, CH₃ -CH₂); 1.25 (m, 1H, β-Leu); 1.39 (m, 1H,--Leu); 1.56 (m, 1H, β'-Leu); 1.98 (dd, J=6.4 Hz, J=13.5 Hz, 1H, β-Phe);2.1-2.4 (m, 2H, CH₂ CH₃); 2.48 (d, J=7.0 Hz, 1H, NH-Phe); 2.77 (dd,J=4.7 Hz, J=13.5 Hz, 1H, β'-Phe); 3.41 (m, 1H, α-Phe); 4.0-4.3 (m, 3H,α-Gly+α'-Gly+α-Leu); 5.20-5.27 (two-d, J=19.9 Hz, 2H, 5--CH₂); 5.41-5.68(two-d, J=17.3 Hz, 2H, 17--CH₂); 6.35 (t, J=5.3 Hz, 1H, NH-Gly);

6.76 (d, J=7.6Hz, 1H, NH-Leu); 6.8-7.3 (m, 21H, 4×(C₆ H₅)+14--H).

Example 4

9-amino-20-O-(L-Phenylalanyl-L-Leucyl-Glycyl camptothecin (7c) ##STR14##

N-trity-L-phenylalanyl-L-leucylglycine p-nitrophenyl ester (8a, 1.14 g,2 mmol), 9-amino-camptothecin (6b, R₁ =NH₂, 0.363 g, 1 mmol),4-dimethylaminopyridine (0.12 g, 1 mmol) were reacted in drydimethylsulfoxide (20 ml) at room temperature as described in Example 3to give the title compound 7c (0.31 g). TLC on Kieselgel plates F₂₄₅(Merck), eluting system chloroform/methanol (9:1 by volume) R_(f) =0.2FD-MS: m/z * M+H!⁺ 682; ¹ H-NMR (400 MHz, CDCl₃) δ: 0.82 (d, J=6.5 Hz,6H, δ-Leu+δ'-Leu); 1.00 (t, J=7.6 Hz, 3H, CH₂ CH₃); 1.25 (m, 1H, δ-Leu);1.41 (m, 1H, -Leu); 1.59 (m, 1H, β'-Leu); 1.99 (dd, J=6.2 Hz, J=13.5 Hz,1H, βPhe); 2.1-2.4 (m, 2H, CH₂ CH₃); 2.47 (d, J=6.5 Hz, 1H, NH-Phe);2.79 (dd, J=4.7 Hz, J=13.5 Hz, 1H, β'-Phe); 3.43 (m, 1H, α-Phe); 4.0-4.3(m, 5H, 9-NH₂ +α-Leu+α-Gly+α'-Gly); 5.04-5.15 (two-d, J=19.9Hz, 2H,CH₂); 5.39-5.66 (two-d, J=17.0 Hz, 2H, 17-CH₂); 6.44 (t, J=5.3 Hz, 1H,NH-Gly); 6.81 (d, J=7.6Hz,

1H, NH-Leu); 6.85 (m, 3H, 10--H + ##STR15## 7.0-7.4 (m, 19H, 3×C₆ H₅##STR16## +14--H); 7.51 (m, 1H, 11--H).

Example 5

Copolymer of N-(2-hydroxypropyl)methacrylamide, 20-O-N-methacryloylglycyl-(6-aminohexanoylyl!camptothecin and N-(2-hydroxy-propyl)methacryloylglycinamide (Al:x=96, , y=3, , z=1) ##STR17##Copolymer of N-(2-hydroxypropyl)methacrylamide and N-methacryloylglycinep-nitrophenylester (0.15 g), prepared as described in Makromol.Chem.,178, 2159 (1977), containing 2.7×10³ equivalents of p-nitrophenylester,was reacted with 20-O-(6-aminohexanoyl)camptothecin (7c, 18 mg),prepared as described in Example 2, in dry dimethylsulfoxide (1 ml) atroom temperature for 18 hours, then with 1-amino-2-propanol (2 μl) forone hour at room temperature. After that, the reaction mixture wastreated with acetone (70 ml). The precipitate was collected, redissolvedwith anhydrous ethanol (5 ml) and reprecipitated with acetone (50 ml) togive the title conjugate Al (0.14 g) containing 5% (w/w) ofcamptothecin. After plasma incubation, conjugate Al releases 10% ofcamptothecin after 120 hours.

Example 6

copolymer of N-(2-hydroxypropyl)methacrylamide, 20-O- N-metha-cryloylglycyl-L-phenylalanyl-L-leucylalycyl!camptothecin andN-(2-hydroxypropyl)methacryloylglycinamide (AP:×=96, y=2.2, z=1.8)##STR18##

Copolymer of N-(2-hydroxypropyl)methylacrylamide andN-N-methacryloylglycine p-nitrophenylester (0.15 g), containing 2.7×10³equivalents of p-nitrophenylester) and 20-O-(Glycyl-L-leucyl-L-phenylalanyl)camptothecin (7b, 20 mg), prepared as described in Example3 were reacted in dry dimethylsulfoxide (1 ml), then with1-amino-2-propanol as described in Example 6 to give the title conjugateA2 (0.14 g), containing 4.8% w/w of camptothecin. After plasmaincubation, conjugate A2 released 100% of camptothecin after 60 hours.

Example 7

Copolymer of N-(² -hydroxypropyl)methacrylamide) 9-amino-20-O-N-methacryloylglycyl-L-phenylalanyl-L-leucylglycyl! campto-thecin andN-(2-hydroxypropyl)methacryloylalycinamide (A3:×=96, y=3, z=1) ##STR19##The title conjugate was prepared by reacting copolymer ofN-(2-hydroxypropyl) methacrylamide and N-methacryloylglycinep-nitrophenylester (0.15 g, containing 2.7×10⁻³ equivalents ofp-nitrophenylester) and9-amino-20-O-(Glycyl-L-leucyl-L-phenylalanyl)camptothecin (7c, 20 mg),prepared as described in Example 4 in dry dimethylsulfoxide (1 ml), thenwith 1-35-amino -2-propanol as described in Example 5. Yield 0.14 g,containing 6.3% w/w of 9-amino-camptothecin. After plasma incubation,conjugate A3 releases 100% of camptothecin after 50 hours.

We claim:
 1. A polymeric conjugate which consists essentially of:(i)from 60 to 99 mol % of N-(2-hydroxypropyl)methacryloylamide units offormula 1: ##STR20## (ii) from 1 to 40 mol % of20-O-(N-methacryloylglycylaminoacyl)camptothecin units of formula 2##STR21## wherein is a spacer group having respective terminal amino andcarbonyl groups which are separated by at least three atoms and O-CPT isa camptothecin, the C-20 hydroxy group of the camptothecin being linkedto the terminal carbonyl group of the spacer group; and (iii) from 0 to10 mol % of N-methylacryloylglycine orN-(2-hydroxy-propyl)methacryloylglycinamide units of formula 3:##STR22## wherein Z is hydroxy or a radical of the formula --NH--CH₂--CH(OH)--CH₃.
 2. The conjugate according to claim 1, wherein the spacergroup is selected from the group consisting of Ala-Gly, Phe-Gly,Phe-Phe, Leu-Gly, Val-Ala, Phe-Ala, Leu-Phe, Leu-Ala, Phe-Leu-Gly,Phe-Phe-Leu, Leu-Leu-Gly, Phe-Tyr-Ala, Phe-Gly-Phe, Phe-Phe-Gly andPhe-Leu-Gly-Phe.
 3. The conjugate according to claim 1, wherein thespacer group has the formula --UN--Y--CO-- wherein Y is a C₃ -C₆ linearor branched alkyl group or Ala-Gly-NH-Y-CO-, Phe-Gly-NH-Y-CO-,Phe-Phe-NH-Y-CO-, Leu-Gly-NH-Y-CO-, Val-Ala-NH-Y-CO-, Phe-Ala-NH-Y-CO-,Leu-Phe-NH-Y-CO-, Leu-Ala-NH-Y-CO-, Phe-Leu-Gly-NH-Y-CO-,Phe-Phe-Leu-NH-Y-CO, Phe-Phe-Leu-NH-Y-CO-, Leu-Leu-Gly-NH-Y-CO-,Phe-Tyr-Ala-NH-Y-CO, Phe-Gly-Phe-NH-Y-CO-, Phe-Phe-Gly-NH-Y-CO-, orPhe-Leu-Gly-Phe-NH-Y-CO-.
 4. The conjugate according to claim 1, whereinO-CPT is a camptothecin of formula 6: ##STR23## wherein R₁ is hydrogen,hydroxy, nitro or amino or a methylenedioxy group bonded to two adjacentcarbon atoms on the A-ring.
 5. The conjugate according to claim 1,wherein from 1 to 10 mol % of the said units of formula 2 are present.6. A conjugate according to claim 1, wherein the camptothecin content isfrom 1 to 10% (w/w).
 7. A process for preparing a polymeric conjugatewhich consists essentially of:(i) from 60 to 99 mol% ofN-(2-hydroxypropyl)methacryloylamide units of formula 1: ##STR24## (ii)from 1 to 40 mol % of 20-O-(N-methacryloylglycylaminoacyl)camptothecinunits of formula 2 ##STR25## wherein is a spacer group having respectiveterminal amino and carbonyl groups which are separated by at least threeatoms and O-CPT is a camptothecin, the C-20 hydroxy group of thecamptothecin being linked to the terminal carbonyl group of the spacergroup; and (iii) from 0 to 10 mol % of N-methylacryloylglycine orN-(2-hydroxy-propyl) methacryloylglycinamide units of formula 3:##STR26## wherein Z is hydroxy or a radical of the formula --NH--CH₂--CH(OH)--CH₃, which process comprises reacting a20-O-acylamino-camptothecin of formula 7:

    H A!--O--CPT                                               (7)

with a polymer consisting essentially of: (iv) from 60 to 99 mol % ofN-(2-hydroxypropyl)methacryloylamide units of formula 1: ##STR27## and(v) from 40 to 1 mol % of N-methyacryloylglycine units of formula 4:##STR28## wherein R₂ is an active ester or (b) hydroxy; and optionallydisplacing the remaining active ester groups with 1 -amino-2-propanol.8. A 20-O-acylamino-camptothecin of formula 7:

    H A!--O--CPT                                               (7)

wherein is a spacer group having respective terminal amino and carbonylgroups which are separated by at least three atoms and O-CPT is acamptothecin, the C--20 hydroxy group of the camptothecin being linkedto the terminal carbonyl group of the spacer group.
 9. A process forpreparing the 20-O-acylamino-camptothecin of formula 7:

    H A!--O--CPT                                               (7)

wherein is a spacer group having respective terminal amino and carbonylgroups which are separated by at least three atoms and O-CPT is acamptothecin, the C--20 hydroxy group of the camptothecin being linkedto the terminal carbonyl group of the spacer group comprising the stepsof: condensing a camptothecin with a N-protected-aminoacyl derivative ofthe formula 8:

    R.sub.3  A!--P                                             (8)

wherein is as above defined, R₃ is an amino-protecting group and P is anactivated ester, in the presence of an activating agent to give aN-protected 20-O-(acylamino) compound having formula 9:

    R.sub.3 -- A!--(O--CPT)                                    (9)

wherein R₃ and are as defined above and O-CPT is a camptothecin, theC--20 hydroxy group of the camptothecin being linked to the terminalcarbonyl group of the spacer group; and removing the N-protecting groupfrom the resulting compound.
 10. A pharmaceutical composition comprisinga pharmaceutically acceptable diluent or carrier, and as activeingredient, a polymeric conjugate as claimed in claim 1.